Promoter and construct for plant transformation

ABSTRACT

The present invention is directed to a promoter, designated MuA. The present invention is also directed to DNA molecules including said promoter, such as a DNA construct comprising the promoter operably linked to one or more genes or antisense DNA. The invention is further directed to transformed plant tissue including the DNA molecule and to transformed plants and seeds thereof. The promoter is useful for driving gene or antisense expression for the purpose of imparting agronomically useful traits such as, but not limited to, increase in yield, disease resistance, insect resistance, herbicide tolerance, drought tolerance and salt tolerance in plants.

This application is a divisional of U.S. patent application Ser. No.09/259,282, filed Mar. 1, 1999 now U.S. Pat. No. 6,222,096.

BACKGROUND OF THE INVENTION

The present invention is directed to a promoter, designated MuA, withless than 80% homology to a promoter of known activity. The constructionof this promoter provides a general method for the discovery of novelsequences with utility as promoters. The present invention is alsodirected to DNA molecules including said promoter, such as a DNAconstruct comprising the promoter operably linked to one or more genesor antisense DNA. The invention is further directed to transformed planttissue including the DNA molecule and to transformed plants and seedsthereof. The promoter is useful for driving gene or antisense expressionfor the purpose of imparting agronomically useful traits such as, butnot limited to, increase in yield, disease resistance, insectresistance, herbicide tolerance, drought tolerance and salt tolerance inplants.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

The selection of a promoter in the genetic engineering of a heterologousgene is often a critical factor in obtaining expression of the desiredgene. Promoters are typically found at the 5′ end of a gene which iseither directly or indirectly recognized and bound by a DNA-dependentRNA polymerase during the initiation of transcription of the gene.Consequently promoters play a major role in regulating gene expression.There are three general classes of promoters used in the geneticengineering of plants: 1) tissue specific promoters, 2) induciblepromoters, and 3) constitutive promoters. Tissue specific or organspecific promoters drive gene expression in a certain tissue such as inthe kernel, root, leaf, or tapetum of the plant. Chemicals orenvironmental stimuli such as heat, cold, wounding, and etc., induceinducible promoters. Although tissue specific and inducible promotersare required for certain applications, constitutive promoters are themost widely used promoters in the industry. Constitutive promoters arecapable of driving a relatively high level of gene expression in most ofthe tissues of a plant. Constitutive promoters are particularly usefulfor producing herbicide tolerant plants. The most widely usedconstitutive promoter in the genetic engineering of plants is the CaMV35S promoter; other constitutive promoters include the maizepolyubiquitin promoter and the rice actin promoter. In general, thosefamiliar with the art agree that constitutive promoters that drive ahigh level of heterologous gene expression in most of the tissues of aplant are few in number and are of considerable value in the field ofgenetic engineering of crop plants.

Thus, it is desired to develop additional constitutive promoters for usein plant transformation.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a strong,constitutive promoter which can effect high level expression of anoperably linked gene or antisense DNA in transformed plants.

Accordingly, in one aspect of the present invention, a novel promoter,designated MuA, comprising a nucleotide sequence as set forth in SEQ IDNO:2 is provided.

In another aspect of the invention, a DNA molecule is provided whichcomprises the MuA promoter operably linked to one or more genes orantisense DNA. The gene or antisense DNA impart an agronomically usefultrait or selectable marker to a transformed plant. In one embodiment,the DNA molecule also includes an additional nucleotide sequence thatinfluences gene expression. In a second embodiment, the DNA molecule ispart of an expression vector. In a third embodiment, the DNA molecule ispart of a transformation vector.

In an additional aspect of the present invention, transformed plantcells and tissues, transformed plants and seeds of transformed plantsare provided. The expression of the gene or antisense DNA is regulatedby the MuA promoter, and, if present, a second regulatory sequence.

In a further aspect of the invention, a method for obtaining novelsequences with utility as promoters is provided. The method involves thereplacement of parts of the sequence of known or newly discoveredpromoters while maintaining or improving the activity of the promoter.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 shows the nucleotide sequence, including all 351 base pairs ofthe “yeast homolog” to the CaMV 35S promoter. The source and GenBankaccession of each homologous sequence from which a given segment ismostly comprised is as follows: A: SC13OKBXV (X94335); B: SCE6592(U18813); C: SCLACHXII (X94607); D: SC40KBRXVJ (S95720); E: YSCH9315(U00059); F: YSCCHRVIN (D50617); G: SCCHRIII (X59720); H: SX9571X(Z49810); I: YSCP9659 (U40829); J: SCYBR223C (Z36092); K: EPFCPCG(M81884); L: YSCF6552A (D31600); M: VSCH9177 (U00029); N: CaMV62(V00141)[CaMV35S-U.S. Pat. No. 532,605]. The three bases that were laterchanged by site directed mutation to that of the CaMV 35S sequence areunderlined.

FIG. 2 shows the MuA promoter sequence (SEQ ID NO:2) which resultedafter site directed mutatgenesis.

FIG. 3 shows the homology between the MuA and the CaMV 35S promotersequences (SEQ ID NO:2 and SEQ ID NO:3, respectively) over a 352 bpoverlap.

FIGS. 4A and 4B presents a comparison of GUS expression resulting fromtransiently expressing the gus gene in plasmids p350096 (FIG. 4A) andpMuA0096 (FIG. 4B).

SUMMARY OF THE SEQUENCES

SEQ ID NO:1 is the sequence of a composite yeast homolog promotersequence. SEQ ID NO:2 is the sequence of the MuA promoter. SEQ ID NO:3is the sequence of a CaMV 35S promoter.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a promoter, designated MuA. Thepresent invention is also directed to DNA molecules including saidpromoter, such as a DNA construct comprising the promoter operablylinked to one or more genes or antisense DNA. The invention is furtherdirected to transformed plant tissue including the DNA molecule and totransformed plants and seeds thereof. The promoter is useful for drivinggene or antisense expression for the purpose of imparting agronomicallyuseful traits such as, but not limited to, increase in yield, diseaseresistance, insect resistance, herbicide tolerance, drought toleranceand salt tolerance in plants.

The present invention is also directed to a general method for obtainingpromoters in which sequences of known effect in a promoter are replacedwith different sequences over their length. It has been found thathybrid promoters can be constructed with over 15% of the originalpromoter sequence replaced. The activity of the original promoter is atleast maintained or improved.

In accordance with the present invention, the promoter MuA isconstructed having less than 85% homology with a promoter of knownactivity. The construction of this promoter provides an example of amore general method for obtaining promoters in which sequences withknown effect are replaced with different sequences over their lengthwhile maintaining or improving the known effect. It has been found thathybrid promoters can be constructed with over 15% of the originalpromoter sequence replaced. This approach is particularly useful withknown constitutive promoters of broad utility in a range of species andwith promoters obtained from species other than the species to begenetically engineered. The success of this approach, as exemplifiedwith the construction of MuA, is surprising in that promoters generallyare thought to be precisely constructed and to be intolerant ofmodifications without substantial loss of activity.

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

A structural gene is a DNA sequence that is transcribed into messengerRNA (mRNA) which is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

A promoter is a DNA sequence that directs the transcription of astructural gene. Typically, a promoter is located in the 5′ region of agene, proximal to the transcriptional start site of a structural gene.If a promoter is an inducible promoter, then the rate of transcriptionincreases in response to an inducing agent. For example, a promoter maybe regulated in a tissue-specific manner such that it is only active intranscribing the associated coding region in a specific tissue type(s)such as leaves, roots or meristem.

In contrast, the rate of transcription is not generally regulated by aninducing agent if the promoter is a constitutive promoter. The promotermay be tissue-general, also known as non-tissue-preferred, such that itis active in transcribing the associated coding region in a variety ofdifferent tissue types.

A core promoter contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity.

An isolated DNA molecule is a fragment of DNA that is not integrated inthe genomic DNA of an organism.

Complementary DNA (cDNA) is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the tern “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand.

To operably link one nucleotide sequence to another refers to joiningtwo DNA fragments to produce a chimeric DNA construct that hasbiological activity. For example, an isolated DNA fragment comprising apromoter, such as the MuA promoter is operably linked to an isolated DNAfragment comprising a structural gene or antisense DNA. The resultingchimeric DNA construct is functional when the MuA promoter is shown toinitiate transcription of the structural gene or antisense DNA.

The term expression refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural genes into mRNA and the translation ofmRNA into one or more polypeptides. In the case of antisense DNA,expression involves transcription of the antisense DNA into an antisenseRNA.

A cloning vector is a DNA molecule, such as a plasmid, cosmid, orbacteriophage, that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance.

An expression vector is a DNA molecule comprising a gene or antisenseDNA that is expressed in a host cell. Typically, gene expression isplaced under the control of certain regulatory elements, includingconstitutive or inducible promoters, tissue-specific regulatoryelements, and enhancers. Such a gene or antisense DNA is said to be“operably linked to” the regulatory elements.

A foreign gene refers in the present description to a DNA sequence thatis operably linked to at least one heterologous regulatory element.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or an expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell.

A transgenic plant is a plant having one or more plant cells thatcontain a structural gene or antisense DNA operably linked to the MuApromoter.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A DNA molecule can be designed tocontain an RNA polymerase II template in which the RNA transcript has asequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an antisense RNA, and a DNA sequence that encodesthe antisense RNA is termed an antisense gene. Antisense RNA moleculesare capable of binding to mRNA molecules, resulting in an inhibition ofmRNA translation.

An antisense nucleic acid (oligonucleotide) is a nucleic acid(oligonucleotide) which has a sequence exactly opposite to an mRNAmolecule made in an organism. Messenger RNA molecules made in anorganism serve as templates for the synthesis of protein. Since the“antisense” mRNA molecule binds tightly to its mirror image, it canprevent a particular protein from being made.

In one aspect of the present invention, a novel promoter, designatedMuA, comprising a nucleotide sequence as set forth in SEQ ID NO:2 isprovided.

In another aspect of the invention, a DNA molecule is provided whichcomprises the MuA promoter operably linked to one or more genes orantisense DNA. The gene or antisense DNA imparts an agronomically usefultrait or selectable marker to a transformed plant. In one embodiment,the DNA molecule also includes an additional nucleotide sequence thatinfluences gene expression. Examples of nucleotide sequences thatinfluence the regulation of heterologous genes include enhancers oractivating regions, such as those derived from CaMV 35S, opine synthasegenes or other plant genes (U.S. Pat. Nos. 5,106,739; 5,322,938;5,710,267; 5,268,526; 5,290,294). In a second embodiment, the DNAmolecule is part of an expression vector. In a third embodiment, the DNAmolecule is part of a transformation vector.

In an additional aspect of the present invention, transformed plantcells and tissues, transformed plants and seeds of transformed plantsare provided. The expression of the gene or antisense DNA is regulatedby the MuA promoter and second regulatory sequence, if present.

By means of the present invention, agronomic genes and selectable markergenes can be operably linked to the MuA promoter and constitutivelyexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Such genes included, but are not limited to, those describedherein.

1. Genes that Confer Resistance to Pests or Disease

(A) Plant Disease Resistance Genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. Examples of such genes include, the tomato Cf-9 genefor resistance to Cladosporium fulvum (Jones et al., 1994), tomato Ptogene, which encodes a protein kinase, for resistance to Pseudomonassyringae pv. tomato (Martin et al., 1993), and Arabidopsis RSSP2 genefor resistance to Pseudomonas syringae (Mindrinos et al., 1994).

(B). A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon, such as, a nucleotide sequence ofa Bt δ-endotoxin gene (Geiser et al., 1986). Moreover, DNA moleculesencoding δ-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), under ATCC accession numbers. 40098, 67136,31995 and 31998.

(C) A lectin, such as, nucleotide sequences of several Clivia miniatamannose-binding lectin genes (Van Damme et al., 1994).

(D) A vitamin binding protein, such as avidin and avidin homologs whichare useful as larvicides against insect pests. See U.S. Pat. No.5,659,026.

(E) An enzyme inhibitor, e.g., a protease inhibitor or an amylaseinhibitor. Examples of such genes include, a rice cysteine proteinaseinhibitor (Abe et al., 1987), a tobacco proteinase inhibitor I (Huub etal., 1993), and an α-amylase inhibitor Sumitani et al., 1993).

(F) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof, such as, baculovirus expression of clonedjuvenile hormone esterase, an inactivator of juvenile hormone (Hammocket al., 1990).

(G) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. Examples of such genesinclude, an insect diuretic hormone receptor (Regan, 1994), anallostatin identified in Diploptera puntata (Pratt, 1989),insect-specific, paralytic neurotoxins (U.S. Pat. No. 5,266,361).

(H) An insect-specific venom produced in nature by a snake, a wasp,etc., such as, a scorpion insectotoxic peptide (Pang, 1992).

(I) An enzyme responsible for a hyperaccumulation of monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(J) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, anuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. Examples ofsuch genes include, a callas gene (PCT published applicationWO93/02197), chitinase-encoding sequences (which can be obtained, forexample, from the ATCC under accession numbers 3999637 and 67152),tobacco hookworm chitinase (Kramer et al., 1993) and parsley ubi4-2polyubiquitin gene (Kawalleck et al., 1993).

(K) A molecule that stimulates signal transduction. Examples of suchmolecules include, nucleotide sequences for mung bean calmodulin cDNAclones (Botella et al., 1994) a nucleotide sequence of a maizecalmodulin cDNA clone (Griess et al., 1994).

(L) A hydrophobic moment peptide. See U.S. Pat. Nos. 5,659,026 and5,607,914, the latter teaches synthetic antimicrobial peptides thatconfer disease resistance.

(M) A membrane permease, a channel former or a channel blocker, such as,a cecropin-β lytic peptide analog (Jaynes et al., 1993) which renderstransgenic tobacco plants resistant to Pseudomonas solanacearum.

(N) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. See,for example, Beachy et al. (1990).

(O) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactive an affected enzyme, killing the insect. Forexample, Taylor et al. (1994) shows enzymatic inactivation in transgenictobacco via production of single-chain antibody fragments.

(P) A virus-specific antibody. See, for example, Tavladoraki et al.(1993), which shows that transgenic plants expressing recombinantantibody genes are protected from virus attack.

(Q) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase (Lamb et al., 1992). The cloningand characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart et al.(1992).

(R) A developmental-arrestive protein produced in nature by a plant,such as, the barley ribosome-inactivating gene have an increasedresistance to fungal disease (Longemann et al., 1992).

2. Genes that Confer Resistance to a Herbicide

(A) A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS (Lee et al., 1988) and AHAS enzyme (Miki et al., 1990).

(B) Glyphosate (resistance imparted by mutant EPSP synthase and aroAgenes, respectively) and other phosphono compounds such as glufosinate(PAT and bar genes), and pyridinoxy or phenoxy proprionic acids andcyclohexones (ACCase inhibitor encoding genes). See, for example, U.S.Pat. No. 4,940,835, which discloses the nucleotide sequence of a form ofEPSP which can confer glyphosate resistance. A DNA molecule encoding amutant aroA gene can be obtained under ATCC accession number 39256, andthe nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061. European patent application No. 0 333 033 and U.S. Pat. No.4,975,374 disclose nucleotide sequences of glutamine synthetase geneswhich confer resistance to herbicides such as L-phosphinothricin. Thenucleotide sequence of a phosphinothricinacetyl-transferase gene isprovided in European application No. 0 242 246. De Greef et al. (1989)describes the production of transgenic plants that express chimeric bargenes coding for phosphinothricin acetyl transferase activity. Exemplaryof genes conferring resistance to phenoxy proprionic acids andcyclohaexones, such as sethoxydim and haloxyfop, are the Acc1-S1,Acc1-S2 and Acc1-S3 genes described by Marshall et al. (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.(1991) describes the use of plasmids encoding mutant psbA genes totransform Chlamydomonas. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648, and DNA molecules containing thesegenes are available under ATCC accession numbers 53435, 67441 and 67442.Cloning and expression of DNA coding for a glutathione S-transferase isdescribed by Hayes et al. (1992).

3. Genes That Confer or Contribute to a Value-Added Trait

(A) Modified fatty acid metabolism, for example, by transforming maizeor Brassica with an antisense gene or stearoyl-ACP desaturase toincrease stearic acid content of the plant (Knultzon et al., 1992).

(B) Decreased Phytate Content

-   -   (1) Introduction of a phytase-encoding gene would enhance        breakdown of phytate, adding more free phosphate to the        transformed plant, such as the Aspergillus niger phytase gene        (Hartingsveldt et al., 1993).    -   (2) A gene could be introduced that reduces phytate content. In        maize, this, for example, could be accomplished, by cloning and        then reintroducing DNA associated with the single allele which        is responsible for maize mutants characterized by low levels of        phytic acid (Raboy et al., 1990).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. Examples of such enzymes include,Streptococcus mucus fructosyltransferase gene (Shiroza et al., 1988),Bacillus subtilis levansucrase gene (Steinmetz et al., 1985), Bacilluslicheniformis α-amylase (Pen et al., 1992), tomato invertase genes(Elliot et al., 1993), barley amylase gene (Søgaard et al., 1993), andmaize endosperm starch branching enzyme II (Fisher et al., 1993).

4. Selectable Marker Genes:

(A) Numerous selectable marker genes are available for use in planttransformation including, but not limited to, neomycinphosphotransferase I, hygromycin phosphotransferase, EPSP synthase anddihydropteroate. See, Miki et al. (1993).

Synthesis of genes suitably employed in the present invention can beeffected by means of mutually priming long oligonucleotides. See, forexample, Ausubel et al. (1990) and Wosnick et al. (1987). Moreover,current techniques which employ the polymerase chain reaction permit thesynthesis of genes as large as 1.8 kilobases in length. See Adang et al.(1993) and Bambot et al. (1993). In addition, genes can readily besynthesized by conventional automated techniques.

In a further aspect of the invention, a method for obtaining novelsequences with utility as promoters is provided. The method involves thereplacement of part of the sequence of known or newly discoveredpromoters while maintaining or improving the activity of the promoters,as demonstrated by the construction of MuA. It has been found thathybrid promoters can be constructed with over 15% of the originalpromoter sequence replaced.

In accordance with the present invention, a novel promoter isconstructed by the following steps. The sequence of a known or newlydiscovered promoter is compared with known nucleic acid sequences, suchas sequences in genomic databases. In one embodiment, this comparison ismade in the GenBank database using a program such as FASTA (GeneticsComputer Group, Madison, Wis.). Additional suitable databases andcomparison programs are known to a person of skill in the art. Segmentsof sequence similar to the query sequence, i.e., the known or newlydiscovered promoter, are identified and selected. Segments areconsidered similar if they have between 60% and 100% sequence identityover the segment being examined. These segments are usually 20–100 basesin length, although smaller or longer segments can also be selected. Theselected sequences are aligned in linear order according to the sequenceof the promoter being modified. The resultant promoter is a hybridpromoter comprised of sequences similar to but different from theoriginal promoter. The short segments that make up the synthetic hybridpromoter may be parts of promoters or regulatory regions from othergenes. The synthetic hybrid promoter is then constructed and empiricallytested in a test expression system to determine its quantitative andqualitative characteristics. If the synthetic hybrid promoter hasmaintained or improved activity, it may be used directly. If thesynthetic hybrid promoter has a lower activity, the sequence of thesynthetic hybrid promoter is further modified by replacing some of thebases to generate a new hybrid promoter. The new hybrid promoter isagain constructed and tested to determine if it has the desiredmaintained or improved activity. This procedure can be performed asoften as necessary to derive the final hybrid promoter having thedesired activity.

The method for developing novel promoters in accordance with the presentinvention is particularly useful in plant biotechnology. There is ageneral lac k of available promoters for use in the production ofcommercially valuable transgenic plants. In the course of constructingtransformation vectors, it is often necessary to use a given promotermultiple times to drive different genes. Those trained in geneticengineering of plants will recognize the undesirability of thiscircumstance as sequence homology within a vector has been associatedwith the phenomenon of gene silencing. By using a synthetic hybridpromoter, prepared in accordance with the present invention, the problemof homology-dependent gene silencing can be ameliorated. In order tomaximize this effect of the hybrid promoter, it is preferred that overat least 15% of the sequence of the original promoter has been replacedin the hybrid promoter.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized. Inthese Examples, corn is used for illustrative purposes only. Other plantspecies are also transformed with the DNA constructs of the presentinvention using techniques well known in the art.

Example 1 Construction of Plasmids used in Corn Transient Assays

A promoter sequence, designated yeast homolog to the CaMV 35S, wasdesigned with around 80% homology to the CaMV 35S promoter (FIG. 1). Thesequence was not randomly derived but based on homologies found in thedatabase at the time of design. Three hundred fifty one bases of thecAMV 35S promoter were compared to plant DNA sequences, which includedsequences from yeast, in the database GenBank using the FASTA program.Regions of homologies were then manually aligned using the CaMV 35Spromoter as the template. A few bases within segments and in regionslinking segments were changed to match the CaMV 35S sequence so that thehomology would remain around 80%. FIG. 1 depicts the yeast homologsequence and the GenBank designations from which the majority of thesequence for a given segment was derived. All of the homologies werefrom yeast with the exception of segment “K” of FIG. 1 which was fromthe chloroplast of Epifagus virginiana and segment “N” of FIG. 1 whichwas from CaMV 35S. The sequence was synthesized by the Molecular BiologyGroup at The Midland Certified Reagent Company, Midland, Tex. with EcoRIand SacI compatible overhangs on the 5′ and 3′ ends, respectively.

Plasmid 350096 was constructed by removing a root preferential promoterwith an EcoRI and SacI double digestion from the plasmid pZM0096 of U.S.Pat. No. 5,633,363 and replacing it with a CaMV 35S promoter frompSLJ4k1 which had compatible EcoRI/SacI cohesive ends. Plasmid pSLJ4k1was obtained from the Sainsbury Laboratory at the John Innes Center,England. Plasmid 350096 consisted of a 1.3 kb CaMV 35S promoter, 0.5 kbintervening sequence six (IV6) from corn alcohol dehydrogenase, 1.8 kbgus reporter gene, 0.25 kb nopaline syntase 3′ end (nos) in a pUC18backbone.

The CaMV 35S promoter in p350096 was replaced with the synthesized yeasthomolog. The plasmid p350096 was digested with EcoRI and SacI, thendephosphorylated to prevent self ligation. The yeast homolog wasdirectionally ligated into the EcoRI and SacI sites to produce pY0096.

The yeast homolog to the CaMV 35S promoter produced a low level oftransient expression when compared to the CaMV 35S promoter in transientexpression experiments in corn callus (see Example 2). An increase inthe level of expression was sought by changing some nucleotides thoughtto be important for the function of the CaMV 35S promoter. Three basesat positions 277, 278, and 279 of the yeast homolog (FIG. 1) werechanged from ACA to CGC by site directed mutagenesis using theQuikChange™ site-directed mutagenesis kit according to the manufacturerStratagene (FIG. 1). This new promoter called MuA (FIG. 2) in plasmidpMuA0096 resulted in a similar level of transient expression in corncallus when compared to the CaMV 35S promoter (see Example 2, FIGS. 4Aand 4B). The sequence of MuA (FIG. 2) has 79.5% homology over 352 baseoverlap to the CaMV 35S promoter published by Gardner et al., 1981. Thecomparison of the MuA promoter with the CaMV 35 S promoter is shown inFIG. 3.

Example 2 Transient Assays in Corn Callus

Tissue preparation: Type II callus of Stine elite inbred 963 was used astarget tissue for transient assays. The Type II callus, initiated fromimmature embryos on N6AMOD medium (Table 1), was maintained as a stockculture on DN62 medium (Table 1). Tissue was transferred every ten days.For transient assays tissue was taken from these cultures (usually 4 to5 days after subculture) and spread on a Whatman No 4 filter disk onDN62OSM medium (Table 1) up to one day prior to bombardment with theparticle inflow gun.

DNA Delivery: A particle inflow gun (PIG) as described by Finer et al.(1992) and Vain et al. (1993) was used to deliver the DNA. In brief, 50mg of tungsten particles (M10 from Sylvania Chemicals/Metals, Towanda,Pa.) were sterilized for 15 minutes in 95% ethanol in a 1.5 ml microfugetube. Particles were rinsed 3 times in sterile distilled water byrepeated vortexing, centrifugation and resuspension in 0.5 ml water.Particle suspensions were made fresh for each experiment. Plasmid DNAwas coated onto the particles by mixing 25 ul of tungsten particlesuspension (2.5 mg), 5 μl of DNA (5 ug), 25 ul of 2.5 M CaCl₂, and 10 ulof 100 mM spermidine (free base). After allowing the particles to settlefor a few minutes while on ice, 50 ul of supernatant was removed. Two ulof the remaining particle suspension was pipetted onto the center of thescreen of a syringe filter unit. The syringe filter unit was reassembledand screwed into the LUER-LOK needle adaptor within the chamber. Thetarget tissue in a petri plate was placed about 15 cm below the syringefilter unit. A vacuum of approximately 28 in Hg was applied and theparticles were discharged when helium (80 psi) was released followingactivation of the solenoid by the timer relay.

TABLE 1 Media Compositions Ingredients/L N6AMOD DN62 DN62OSM N6 salts¹3.98 g 3.98 g 3.98 g N6 vitamins² 1 ml 1 ml 1 ml Asparagine 150 mg 800mg 800 mg Myo-inositol 100 mg 100 mg 100 mg Proline 700 mg 1400 mg 1400mg Casamino Acids 100 mg 100 mg 2,4-D 1 mg 1 mg 1 mg Sucrose 20 g 20 g20 g Glucose 10 g MES 500 mg Mannitol 45.5 g Sorbitol 45.5 g AgNO₃ 10 mgGelrite 2 g 3 g 3 g pH 6.0 5.8 5.8 ¹N6 salts - Sigma Plant CultureCatalogue ref. C 1416. ²N6 vitamins: 2 mg/l glycine, 0.5 mg/l nicotinicacid, 0.5 mg/l pyridoxine HCl, 1 mg/l thiamine HCl (Chu, 1978).

After bombardment, callus was incubated at 250° C. in the dark for 16–24h on the same medium used for bombardment. Then, transient gusexpression was evaluated by incubating the tissue in 0.5 mg/ml X-gluc(Gold Biotechnology, Inc. St. Louis, Mo.) in 0.1M sodium phosphatebuffer pH 7.0 and 0.1% TRITON-X-100 at 37° C. for 4–16 h after which thenumber and intensity of blue foci were evaluated under a stereomicroscope at approximately 10× magnification. Tissue was transformedwith either p350096, or pMuA0096. Results are shown in FIGS. 4A and 4B.It is seen that tissue transformed with p350096 (FIG. 4A) or pMuA0096(FIG. 4B) had similar levels of transient expression. Tissue transformedwith pY0096 was found to have lower levels of transient expression.

Example 3 Construction of Plasmids Used in the Transformation of Corn

The bar gene in pBARGUS (Fromm et al., 1990) was used to replace the gusgene in pMuA0096 using the BamHI and PstI sites. Briefly, pBARGUS wasdigested with BamHI and PstI and the 800 bp fragment containing the bargene was isolated. The plasmid pMuA0096 was digested with BamHI and PstIand the ends were dephosphorylated. The 800 bp fragment containing thebar gene was ligated to the digested, dephosphorylated pMuA0096 toproduce the plasmid pMuABar.

The MuABar expression cassette was removed from pMuABar by digestionwith EcoRI and HindIII and cloned into pSB11 (obtained from JapanTobacco) after digestion with EcoRI and HindIII and dephosphorylation,resulting in the plasmid pSB11MuABar. Plasmid pSB11MuABar was combinedwith pSB1 (obtained from Japan Tobacco) via homologous recombination andmobilized into Agrobacterium LBA 4404 via triparental mating accordingto U.S. Pat. No. 5,591,616 resulting in pSBMuABar. This plasmid in LBA4404 was used in the transformation of corn.

Example 4 Transformation of Corn

Agrobacterium-mediated DNA delivery was used to produce stable corntransformants carrying MuA driving the bar gene. Agrobacterium LBA 4404harboring PSB11MuABar recombined with pSB1 (Example 3) was taken fromglycerol stocks and streaked out on YP medium (5 g/l yeast extract, 10g/l peptone, 5 g/l NaCl, 15 g/l agar and pH 6.8) supplemented with 50mg/l spectinomycin and grown for one or two days at 28° C.

Immature embryos of Stine elite inbred 963 were aseptically removed fromkernels of plants grown in a grow room (15 h photoperiod, 28° day and25° night). Embryos were harvested 10 to 11 days after pollination whenthey were between 1 mm and 2 mm in length and then placed in 2 ml ofLSinf medium (Table 2) in an Eppendorf tube. The mixture was thenstirred with a vortex mixer (VORTEX GENIE 2) at full speed for 5seconds, the LSinf removed, replaced with fresh medium and then stirredagain. All medium was then removed from the tube using a Pasteurpipette. Bacteria were collected with a platinum loop (enough to coatthe wire of the loop) and thoroughly suspended in 1 ml of Lsinf-ASmedium (Table 2) using a Pasteur pipette. The bacterial suspension wasthen introduced into the tube containing the embryos and the mixturestirred with a vortex mixer at full speed for 30 seconds. After this theembryos were allowed to stand for five minutes and were then transferredto the surface of LSAS medium (Table 2) solidified with agar, care beingtaken to remove any accompanying liquid. Embryos were immediatelyoriented so that the scutellar surface was uppermost.

TABLE 2 Media Compositions Ingredients/L LSAS Lsinf LsinfAS MSsalts/vits¹ 4.43 g 4.43 g 4.43 g Proline 700 mg Casamino Acids 1.0 g 1.0g Na₂EDTA 37.3 mg 37.3 mg 37.3 mg 2,4-D 1.5 mg 1.5 mg 1.5 mg MES 500 mgThiamine HCl 1.0 mg 1.0 mg Sucrose 20 g 68.5 g 68.5 g Glucose 10 g 36.0g 36.0 g Acetosyringone 100 um 100 um Phytagar 7 g pH 5.8 5.2 5.2 ¹MSsalts/vits - Sigma Plant Culture Catalogue ref. M5519

The embryos were then cultured in the dark at 19° for 48 hours. Afterthis time the plates were removed from the incubator and placed at 45°for 30 minutes. Then they were returned to the 19° incubator for afurther day. Following this the embryos were transferred to DN62ALC(Table 3) and incubated at 24° for 5 days. Next, the embryos weretransferred to DN62ALCB (Table 3) and incubated at 24° for 14 days. Forthe next 14-day passage the cefotaxime concentration was raised from 50mg/l (in DN62ALC) to 250 mg/l. This medium—DN62ACB (Table 3)—allowed fora better control of the residual Agrobacterium cells contaminating thecorn embryos. Embryos were then transferred back to DN62ALCB for afurther 14 days. At this time, transformed corn clones could berecognized by their ability to grow as prolific Type II callus on thebialaphos-containing medium. Culture of the clones continued on DN62Bmedium (see Table 3) for a further two weeks after which time the TypeII callus was transferred to DNROB medium (Table 4) to initiateregeneration. After one to two weeks on DNROB somatic embryos developedas individual structures. These embryos were allowed to mature for oneto two weeks on a further passage of DNROB (Table 4) and were thentransferred to DNO6S (Table 4). Finally, they were transferred to MSOGor 1/2MS0.1IBA (Table 4) where they germinated and formed plantlets. Theplantlets were then transferred to tubes containing 1/2MS 0.1IBA whereroots developed. The plants were transferred to peat pots prior to goinginto the greenhouse. In the greenhouse the plants were grown to maturityand seed collected either after backcrossing to Stine inbred 963 orafter selfing.

The presence of an expressing bar gene was then confirmed by leafpainting with Liberty, both in the primary transformants and in progeny.Mendelian ratios of an expressing bar gene were routinely observed inthe progeny.

TABLE 3 Media Compositions Ingredients/L DN62B DN62ALC DN62ALCB DN62ACBN6 salts¹ 3.98 g 3.98 g 3.98 g 3.98 g N6 vitamins¹ 1 ml 1 ml 1 ml 1 mlAsparagine 800 mg 800 mg 800 mg 800 mg Myo-inositol 100 mg 100 mg 100 mg100 mg Proline 1400 mg 1400 mg 1400 mg 1400 mg Casamino Acids 100 mg 100mg 100 mg 100 mg 2,4-D 1 mg 1 mg 1 mg 1 mg Sucrose 20 g 20 g 20 g 20 gGlucose 10 g AgNO₃ 10 mg 10 mg 10 mg Bialaphos 1 mg 1 mg 1 mg Cefotaxime50 mg 50 mg 250 mg Gelrite 3 g 3 g 3 g 3 g pH 5.8 5.8 5.8 5.8 ¹N6 saltsand vitamins as in Table 1.

TABLE 4 Media Compositions 1/2MS Ingredients/L DNROB DNO6S MSOG 0.1IBAMS Salts¹ 4.43 g 4.43 g 4.43 g 2.215 g Asparagine 800 mg Proline 1400 mgNa₂EDTA 37.3 mg 37.3 mg 37.3 mg 37.3 mg Casamino Acids 100 mg NicotinicAcid 0.5 mg Gibberellic Acid 0.1 mg Indole-3-Butyric Acid 0.1 mg Sucrose60 g 30 g 20 g Sorbitol 20 g Bialaphos 1 mg Gelrite 2 g 2 g Phytagar 7 g7 g pH 5.8 5.8 5.8 5.8 ¹MS Salts - Sigma Plant culture Catalogue ref.M5519

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

LIST OF REFERENCES

-   Abe et al. (1987). J. Biol. Chem. 262:16793.-   Adang et al. (1993), Plant Molec. Biol. 21:1131.-   Ausubel et al. (1990). Current Protocols in Molecular Biology, Wiley    Interscience, pp. 8.2.8–8.2.13.-   Bambot et al. (1993). PCR Methods and Applications 2:266.-   Beachy et al. (1990). Ann. Rev. Phytopathol. 28:451.-   Botella et al. (1994). Plant Molec. Biol. 24:757.-   Chu C. C. (1978). “The N6 medium and its application to anther    culture of cereal crops.” In Proc. Symp. on Plant Tissue Culture.    Sci. Press, Beijing, pp 43–50.-   De Greef et al. (1989). Bio/Technology 7:61.-   Elliot et al. (1993). Plant Molec. Biol. 21:515.-   Finer, J. J. et al. (1992). Plant Cell Reports 11:323–328.-   Fisher et al. (1993). Plant Physiol. 102:1045.-   Fromm, et al. (1990). Biotechnology 8:833–839.-   Gardner et al. (1991). Nucl. Acids Res. 9:2871–2888.-   Geiser et al. (1986). Gene 48:109.-   Griess et al. (1994). Plant Physiol. 104:1467.-   Hammock et al. (1990). Nature 344:458.-   Hayes et al. (1992). Biochem. J. 285:173.-   Huub et al. (1993). Plant Molec. Biol. 21:985.-   Jaynes et al. (1993). Plant Sci. 89:43.-   Jones et al. (1994). Science 266:789.-   Kawalleck et al. (1993). Plant Molec. Biol. 21:673.-   Knultzon et al. (1992). Proc. Nat. Acad. Sci. USA 89:2624.-   Kramer et al. (1993). Insect Molec. Biol. 23:691.-   Lamb et al. (1992). Bio/Technology 10:1436.-   Lee et al. (1988). EMBO J. 7:1241.-   Longemann et al. (1992). Bio/Technology 10:3305.-   Marshall et al. (1992). Theor. Appl. Genet. 83:435.-   Martin et al. (1993). Science 262:1432.-   Miki et al. (1990). Theor. Appl. Genet. 80:449.-   Miki et al. (1993). “Procedures for Introducing Foreign DNA into    Plants,” in Methods in Plant Molecular Biology and Biotechnology,    Glick et al. (eds.), CRC Press, pp. 67–88.-   Mindrinos et al. (1994). Cell 78:1089.-   Pang et al. (1992). Gene 116:165.-   Pen et al. (1992). Bio/Technology 10:292.-   Pratt et al. (1989). Biochem. Biophys. Res. Comm. 163:1243.-   Przibilla et al. (1991). Plant Cell 3:169.-   Raboy et al. (1990). Maydica 35:383.-   Regan (1994). J. Biol. Chem. 269:9.-   Shiroza et al. (1988). J. Bacteriol. 170:810.-   Søgaard et al. (1993). J. Biol. Chem. 268:22480.-   Steinmetz et al. (1985). Mol. Gen. Genet. 200:220.-   Sumitani et al. (1993). Biosci. Biotech. Biochem. 57:1243.-   Tavladoraki et al. (1993). Nature 266:469.-   Taylor et al. (1994). Abstract #497, Seventh Int'l. Symposium on    Molecular Plant-Microbe Interactions.-   Toubart et al. (1992). Plant J. 2:367.-   Vain, P. et al. (1993). Plant Cell, Tissue and Organ Culture    33:237–246.-   Van Damme et al. (1994). Plant Molec. Biol. 24:825.-   Van Hartingsveldt et al. (1993). Gene 127:87.-   Wosnick et al. (1987). Gene 60:115.-   U.S. Pat. No. 4,769,061.-   U.S. Pat. No. 4,810,648.-   U.S. Pat. No. 4,940,835.-   U.S. Pat. No. 4,975,374.-   U.S. Pat. No. 5,106,739.-   U.S. Pat. No. 5,266,317.-   U.S. Pat. No. 5,268,526.-   U.S. Pat. No. 5,290,294.-   U.S. Pat. No. 5,322,938.-   U.S. Pat. No. 5,591,616.-   U.S. Pat. No. 5,607,914.-   U.S. Pat. No. 5,633,363.-   U.S. Pat. No. 5,659,026.-   U.S. Pat. No. 5,710,267.-   PCT published application No. WO 93/02197.-   European published application No. 0 242 246.-   European published application No. 0 333 033.

1. A method for preparing a synthetic promoter which comprises the stepsof: a) comparing the sequence of a template promoter with known nucleicacid sequences selected from a GenBank database or an equivalentdatabase; b) selecting 20 base pair to 100 base pair segments of saidknown nucleic acid sequences selected from a GenBank database or anequivalent database that have between 60% and 100% homology to 20 basepair to 100 base pair segments of the template promoter sequence; c)placing said 20 base pair to 100 base pair segments in the order inwhich said segments occur in the template promoter to produce a firstsynthetic promoter having over at feast 15% of its sequence differentfrom that of the template promoter; and d) testing the first syntheticpromoter for activity.
 2. The method of claim 1, which further comprisesthe steps of: e) modifying the sequence of the first synthetic promoterwhich does not have maintained or improved activity compared to thetemplate promoter to produce a second synthetic promoter; and f) testingthe synthetic promoter for activity.
 3. The method of claim 2, whereinsteps (e) and (f) are repeated one or more times until a syntheticpromoter is produced which has maintained or improved activity comparedto the template promoter.